Virulent M. tuberculosis induces life-long immunity against reinfection in individuals who develop the latent form of this disease. The resulting immunity is substantially better than that achieved in BCG vaccinated animals (and presumably, people). Virulent M. tb H37Rv was attenuated by a targeted mutagenesis of the leu and lys genes and the resulting auxotrophs were injected into mice and guinea pigs to assess their survival in the lung and spleen as a measure of their residual virulence and immunogenicity. The mice were challenged with a small aerogenic inoculum of M. tb Erdman 1-3 months later and the growth in the lungs was compared with that for the BCG controls. Auxotrophic H37RvLeu- induced high levels of anti-tuberculous resistance in vaccinated C57BL/6 mice which was equivalent or better than that seen in BCG controls. However, mice infected with large numbers of H37RvLeu- showed some mortality and tests carried out on the auxotroph indicated an unacceptable level of back mutation to the wild type. Studies with this auxotroph was discontinued. Tests with lysine auxotrophs of BCG Pasteur and Erdman were initiated in C57BL/c mice. The auxotrophs failed to multiply invivo and did not induce detectable anti-tuberculous resistance to an aerogenic Erdman challenge. However, mice which received two doses of the lysine auxotroph did express an enhanced resistance to challenge, although this was less than that observed in the BCG vaccinated controls. These experiments are continuing.